PROJECT SUMMARY Mother-to-child transmission (MTCT) results in nearly a quarter-million new infant HIV-1 infections each year, mostly occurring in resource limited countries. The long-term goal is to meet the critical need to develop immune interventions to cure infant HIV-1 infection resulting from MTCT. One novel potential strategy is the use of antibody (Ab)-based immunotherapy. Recently developed bispecific Ab-based Dual Affinity Re- Targeting proteins (DARTs) are an innovative type of immunotherapeutic protein capable of recruiting polyclonal adult CD8+ T cells to eliminate HIV-1 infected, and reactivated latently-infected, autologous CD4+ T cells. However, there is a gap in knowledge regarding the ability of DARTs to recruit and redirect newborn infant immune effector cells to target cells infected with HIV-1 virus isolates involved in MTCT. The overall objective of this proposal is to use umbilical cord blood as a model of the nave newborn immune system to identify DARTs or combinations of DARTs that can recruit neonatal cytolytic effector cells to kill autologous CD4+ T cells infected with infant transmitted/founder virus isolates, and reactivated latent virus. The central hypothesis of this proposal is that DARTs can be used to recruit and redirect polyclonal neonatal CD8+ T cells and natural killer (NK) cells for eradication of cells infected with viruses responsible for MTCT. The specific aims of the proposal are 1) Quantify the ability of HIV-specific DARTs to redirect cord blood CD8+ T cells to kill autologous CD4+ T cells infected with infant T/F virus isolates; 2) Enhance DART-mediated killing of HIV-1 infected cord blood CD4+ T cells; and 3) Quantify the ability of HIV-specific DARTs to eradicate reactivated latently-HIV-infected cells in a cord blood CD4+ T cell latency model. The rationale for the proposed research is that newborn infant infection resulting from perinatal or postnatal MTCT likely represents the most favorable context for successful cure of HIV-1 using passive immunotherapy because cost is mitigated by low infant body weight, and therapy can be initiated within days to weeks of the transmission event; prior to the establishment of large populations latently infected cells. Due to phenotypic and functional differences in effector cell populations present in newborn infants and adults it is crucial to perform evaluation of infant passive immunotherapies with model systems that use cells isolated from umbilical cord blood to recapitulate the effector cells present in newborn infants. In the applicant's opinion the approach is innovative because of the use of novel HIV-specific T cell and NK cell recruiting DARTs, and the development of cord blood model systems to evaluate killing of infected cells and reactivated latently infected cells. The results obtained by completion of the aims of this proposal will be significant because they will demonstrate the efficacy of DARTs for elimination of infant cells infected with HIV-1 T/F virus, and will provide the data needed to support the development of DART-based passive immunization strategies designed to cure infant HIV-infection.